Difference between revisions of "Part:BBa K2052018"
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<partinfo>BBa_K2052018 short</partinfo>
 
<partinfo>BBa_K2052018 short</partinfo>
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==Usage & Biology ( Description )==
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This composite part consists of RBS , a coding region ( ButCoAT ) and enables  E.coli to secrete ButCoAT.
  
 
ButCoAT is an enzyme (Butyryl-CoA:acetate CoA-transferase) (EC 2.8.3.8) (Uniprot:C7GB37) which converts Acetyl-CoA (Acetyl-CoA is readily present in the cytosol and it can be directly converted into Butyrate) into Butyrate and can be found in Roseburia intestinalis L1-82. The equation of the reaction (acyl-CoA:acetate CoA-transferase) is given down below :
 
ButCoAT is an enzyme (Butyryl-CoA:acetate CoA-transferase) (EC 2.8.3.8) (Uniprot:C7GB37) which converts Acetyl-CoA (Acetyl-CoA is readily present in the cytosol and it can be directly converted into Butyrate) into Butyrate and can be found in Roseburia intestinalis L1-82. The equation of the reaction (acyl-CoA:acetate CoA-transferase) is given down below :
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Figure 2: Crystal Structures of Acetobacter aceti Succinyl (Butyryl)-CoA:Acetate CoA-Transferase Reveal Specificity Determinants and Illustrate the Mechanism Used by Class I CoA-Transferases.(Mullins, E.A. et al., 2012)
 
Figure 2: Crystal Structures of Acetobacter aceti Succinyl (Butyryl)-CoA:Acetate CoA-Transferase Reveal Specificity Determinants and Illustrate the Mechanism Used by Class I CoA-Transferases.(Mullins, E.A. et al., 2012)
  
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===Butyrate===
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RBS and our protein coding region (ButCoaT) are digested from our whole construct with the enzymes SpeI, Sall, Xbal.
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Butyrate is a short chain fatty acid which is formed by bacterial fermentation of carbohydrates (Sengupta et al., 2005).  
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 +
===Cloning===
 +
 
 +
==Gel Results==
 +
 
 +
[[File:ButCoAT Gel.jpeg|900px]]
 +
 
 +
We have loaded an uncut version of K2052015 next to an EcoRI single digested one. In the third lane RBS ligated ButCoat (K2052018) was loaded with its single digested version. Digested ones gave a sharp lane at 3600 bp as we expected.
 +
 
 +
==Characterization==
 +
 
 +
 
 +
 
 +
==Ligated Parts Transformation Results==
 +
 
 +
Plate resmi var o eklenecek
 +
Resim 8
 +
 
 +
Figure: After ligating ButcoaT and RFP, we have  obtain colonies 2:1 (insert:vector) ratio, and transform them into E.coli BL21.
 +
 
 +
==Confirmational PCR Result==
 +
 
 +
resim 9 eklenecek
 +
 
 +
Figure: Constitutive promoter-RBS-ButCoaT-GFP-Double terminator cloned into pSB1C3 and after ligation we have transformed into E.coli BL21. Colony PCR results here you can see; one primer stick on insert and another stick on vector. The expected  lenght of product is around 850 bp.
 +
 
 +
 
 +
==Flowcytometry Result==
 +
 
 +
After checking with confirmational PCR we have tried to validate RFP fluoresence experimenttaly through Fluorescence Microscope, Here you can see the analysis.
 +
 
 +
 
 +
 
 +
Figure: After measuring RFP signals with channel FL3 we have obtained these graphs. Here again upper triplet stand for Mock(reference point) analysis that is the bacterial culture transformed with only ButCoaT. Middle triplet stand  for only RFP expressed bacterial culture analysis which gave a peak that could be used as a control group. The bottom triplet was RFP tagged ButCoaT expressed bacterial group and as we have shown there is any signal depend on RFP activity.
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 +
 
 +
 
  
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Butyrate putatively suppresses colorectal cancer (CRC). It induces apoptosis and differentiation, inhibits proliferation of tumorous cells in colon flora. At the intestinal level, butyrate plays an administrative part on the transepithelial liquid transport, improves mucosal aggravation and oxidative status, strengthens the epithelial protection obstruction, and regulates instinctive affectability and intestinal motility.
 
  
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==Modeling==
 
  
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[[File:ButCoaTModeling.jpeg|none|700px|caption]]
 
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The figure above shows the increase in ButCoAT molecules over a time span of 10 seconds.
 
−
[[File:ButyrateModeling.jpeg|none|700px|caption]]
 
−
The figure above shows the molar increase in Butyrate concentration over a time span of 10 seconds.
 
  
 
==Design Notes==
 
==Design Notes==
 +
RBS and our protein coding region (ButCoaT) are digested from our whole construct with the enzymes SpeI, Sall, Xbal.
 +
 +
For ButCoaT ;
 +
E.Coli is synthesised suitable for Codon Bias.
 +
In the expression of pet28a (expression vector) , stop codon is removed in order to add HisTag.
 +
 +
==Modelling==
 +
 +
[[File:ButCoAT_graph.jpeg|900px]]
 +
 +
The figure above shows the increase in ButCoAT molecules over a time span of 10 seconds.
 +
 +
[[File:Butyrate graph.jpeg|900px]]
 +
 +
The figure above shows the molar increase in Butyrate concentration over a time span of 10 seconds.
 +
 +
=Source=
 +
The ribosome binding site (B0034) that we’ve used is gotten from iGEM part library.
 +
Source of ButCoaT : Roseburia intestinalis L1-82
 +
 +
===References===
 +
 +
Hassig, C.A., Tong, J.K., Schreiber, S.L. (1997). Fiber-derived Butyrate and the Prevention of Colon Cancer
 +
 +
Roberto Berni Canani, Margherita Di Costanzo, Ludovica Leone, Monica Pedata, Rosaria Meli, Antonio Calignano. (2011). Potential beneficial affects of butyrate in intestinal and extraintestinal diseases
 +
 +
Duncan,S. H., Barcenilla, A., Stewart, C. S., Pryde, S. E., Flint, H. J. (2002). Acetate Utilization and Butyryl Coenzyme A (CoA):Acetate-CoA Transferase in ButyrateProducing Bacteria from the Human Large Intestine
 +
 +
Sequence : http://www.uniprot.org/uniprot/C7GB37?sort=score
 +
 +
Pathway : http://www.genome.jp/dbget-bin/www_bget?reaction+R00393+R01179+R01359+R07832
  
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E.coli is synthesized suitable for Codon Bias.
+
Picture : http://www.brenda-enzymes.org/structure.php?show=reaction&id=360609&type=S&displayType=marvin
  
−
In the expression of pet28a (expression vector) , stop codon is removed for the addition of  HisTag.
 
  
−
==DNA Gel Analysis==
 
  
−
===Characterization===
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PCR Conformation of ButCoAT + pSB1C3
+
RBS and our protein coding region (ButCoaT) are digested from our whole construct with the enzymes SpeI, Sall, Xbal.  
−
[[File:ButCoaTGelImage.jpeg|none|500px|]]
+
−
We have loaded the uncut version of K2052015 (RBS + ButCoAT) next to EcoRI single digested one. In the third lane, RBS ligated ButCoAT (K2052018) was loaded with its single digested version. Digested ones gave sharp lane at 3600 bp as we expected.
+
  
−
==Source==
+
For ButCoaT ;
  
−
We obtained the enzyme ButCoAT from Roseburia intestinalis L1-82 by doing codon bias to our E.coli.
+
E.Coli is synthesised suitable for Codon Bias.
 +
In the expression of pet28a (expression vector) , stop codon is removed in order to add HisTag.  
  
 +
==Source==
 +
The ribosome binding site (B0034) that we’ve used is gotten from iGEM part library.
 +
Source of ButCoaT : Roseburia intestinalis L1-82
  
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<!-- -->
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References
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2052018 SequenceAndFeatures</partinfo>
+
  
 +
Hassig, C.A., Tong, J.K., Schreiber, S.L. (1997). Fiber-derived Butyrate and the Prevention of Colon Cancer
  
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<!-- Uncomment this to enable Functional Parameter display
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Roberto Berni Canani, Margherita Di Costanzo, Ludovica Leone, Monica Pedata, Rosaria Meli, Antonio Calignano. (2011). Potential beneficial affects of butyrate in intestinal and extraintestinal diseases
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===Functional Parameters===
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<partinfo>BBa_K2052018 parameters</partinfo>
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−
<!-- -->
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==References==
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Duncan,S. H., Barcenilla, A., Stewart, C. S., Pryde, S. E., Flint, H. J. (2002). Acetate Utilization and Butyryl Coenzyme A (CoA):Acetate-CoA Transferase in ButyrateProducing Bacteria from the Human Large Intestine
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Weinstock G., Sodergren E., Clifton S., Fulton L., Fulton B., Courtney L., Fronick C., Harrison M., Strong C., Farmer C., Delahaunty K., Markovic C., Hall O., Minx P., Tomlinson C., Mitreva M., Nelson J., Hou S.  Wilson R.K. (2009 , August ). UniProtKB - C7GB37 (C7GB37_9FIRM). Retrieved from http://www.uniprot.org/uniprot/C7GB37?sort=score
+
  
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Sengupta, S., Muir, G.J., Gibson, P. R. (2005). Does Butyrate Protect from Colorectal Cancer?
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Sequence : http://www.uniprot.org/uniprot/C7GB37?sort=score
  
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Figure 1 ;
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Pathway : http://www.genome.jp/dbget-bin/www_bget?reaction+R00393+R01179+R01359+R07832
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BRENDA. Reaction catalyzed by acetate CoA-transferase (2.8.3.8). Retrieved from http://www.brenda-enzymes.org/structure.php?show=reaction&id=360609&type=S&displayType=marvin
+
  
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Figure 2 ;
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Picture : http://www.brenda-enzymes.org/structure.php?show=reaction&id=360609&type=S&displayType=marvin
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SWISS-MODEL. Succinyl-CoA:acetate CoA-transferase (AarCH6-S71A) in complex with CoA. Retrieved from https://swissmodel.expasy.org/templates/4eu8.1
+

Revision as of 21:45, 19 October 2016


Butyryl-CoA:acetate CoA-transferase


Usage & Biology ( Description )

This composite part consists of RBS , a coding region ( ButCoAT ) and enables E.coli to secrete ButCoAT.

ButCoAT is an enzyme (Butyryl-CoA:acetate CoA-transferase) (EC 2.8.3.8) (Uniprot:C7GB37) which converts Acetyl-CoA (Acetyl-CoA is readily present in the cytosol and it can be directly converted into Butyrate) into Butyrate and can be found in Roseburia intestinalis L1-82. The equation of the reaction (acyl-CoA:acetate CoA-transferase) is given down below :

Pathwayson.png

Figure 1: Butanoyl-CoA + Acetate <=> Butanoic acid + Acetyl-CoA

ButCoaTStructure.gif

Figure 2: Crystal Structures of Acetobacter aceti Succinyl (Butyryl)-CoA:Acetate CoA-Transferase Reveal Specificity Determinants and Illustrate the Mechanism Used by Class I CoA-Transferases.(Mullins, E.A. et al., 2012)

RBS and our protein coding region (ButCoaT) are digested from our whole construct with the enzymes SpeI, Sall, Xbal.

Cloning

Gel Results

ButCoAT Gel.jpeg

We have loaded an uncut version of K2052015 next to an EcoRI single digested one. In the third lane RBS ligated ButCoat (K2052018) was loaded with its single digested version. Digested ones gave a sharp lane at 3600 bp as we expected.

Characterization

Ligated Parts Transformation Results

Plate resmi var o eklenecek Resim 8

Figure: After ligating ButcoaT and RFP, we have obtain colonies 2:1 (insert:vector) ratio, and transform them into E.coli BL21.

Confirmational PCR Result

resim 9 eklenecek

Figure: Constitutive promoter-RBS-ButCoaT-GFP-Double terminator cloned into pSB1C3 and after ligation we have transformed into E.coli BL21. Colony PCR results here you can see; one primer stick on insert and another stick on vector. The expected lenght of product is around 850 bp.


Flowcytometry Result

After checking with confirmational PCR we have tried to validate RFP fluoresence experimenttaly through Fluorescence Microscope, Here you can see the analysis.


Figure: After measuring RFP signals with channel FL3 we have obtained these graphs. Here again upper triplet stand for Mock(reference point) analysis that is the bacterial culture transformed with only ButCoaT. Middle triplet stand for only RFP expressed bacterial culture analysis which gave a peak that could be used as a control group. The bottom triplet was RFP tagged ButCoaT expressed bacterial group and as we have shown there is any signal depend on RFP activity.




Design Notes

RBS and our protein coding region (ButCoaT) are digested from our whole construct with the enzymes SpeI, Sall, Xbal.

For ButCoaT ; E.Coli is synthesised suitable for Codon Bias. In the expression of pet28a (expression vector) , stop codon is removed in order to add HisTag.

Modelling

ButCoAT graph.jpeg

The figure above shows the increase in ButCoAT molecules over a time span of 10 seconds.

Butyrate graph.jpeg

The figure above shows the molar increase in Butyrate concentration over a time span of 10 seconds.

Source

The ribosome binding site (B0034) that we’ve used is gotten from iGEM part library. Source of ButCoaT : Roseburia intestinalis L1-82

References

Hassig, C.A., Tong, J.K., Schreiber, S.L. (1997). Fiber-derived Butyrate and the Prevention of Colon Cancer

Roberto Berni Canani, Margherita Di Costanzo, Ludovica Leone, Monica Pedata, Rosaria Meli, Antonio Calignano. (2011). Potential beneficial affects of butyrate in intestinal and extraintestinal diseases

Duncan,S. H., Barcenilla, A., Stewart, C. S., Pryde, S. E., Flint, H. J. (2002). Acetate Utilization and Butyryl Coenzyme A (CoA):Acetate-CoA Transferase in ButyrateProducing Bacteria from the Human Large Intestine

Sequence : http://www.uniprot.org/uniprot/C7GB37?sort=score

Pathway : http://www.genome.jp/dbget-bin/www_bget?reaction+R00393+R01179+R01359+R07832

Picture : http://www.brenda-enzymes.org/structure.php?show=reaction&id=360609&type=S&displayType=marvin



RBS and our protein coding region (ButCoaT) are digested from our whole construct with the enzymes SpeI, Sall, Xbal.

For ButCoaT ;

E.Coli is synthesised suitable for Codon Bias. In the expression of pet28a (expression vector) , stop codon is removed in order to add HisTag.

Source

The ribosome binding site (B0034) that we’ve used is gotten from iGEM part library. Source of ButCoaT : Roseburia intestinalis L1-82

References

Hassig, C.A., Tong, J.K., Schreiber, S.L. (1997). Fiber-derived Butyrate and the Prevention of Colon Cancer

Roberto Berni Canani, Margherita Di Costanzo, Ludovica Leone, Monica Pedata, Rosaria Meli, Antonio Calignano. (2011). Potential beneficial affects of butyrate in intestinal and extraintestinal diseases

Duncan,S. H., Barcenilla, A., Stewart, C. S., Pryde, S. E., Flint, H. J. (2002). Acetate Utilization and Butyryl Coenzyme A (CoA):Acetate-CoA Transferase in ButyrateProducing Bacteria from the Human Large Intestine

Sequence : http://www.uniprot.org/uniprot/C7GB37?sort=score

Pathway : http://www.genome.jp/dbget-bin/www_bget?reaction+R00393+R01179+R01359+R07832

Picture : http://www.brenda-enzymes.org/structure.php?show=reaction&id=360609&type=S&displayType=marvin